Pseudomonas aeruginosa (PA) is a virulent bacterial pathogen that is associated with both acute and chronic lung infections. A group of key virulence factors is the type III secretion system (TTSS), a contact- dependent "molecular syringe" that allows the direct injection of effector molecules from the bacterial cytoplasm into the host cell cytoplasm. While most of the damage is likely caused by the translocated effectors, studies in cell culture and animal models of PA infection indicate that the insertion of TTSS needle apparatus itself is cytotoxic to eukaryotic cells and plays a role in virulence. In this grant, I propose to use RNAi-mediated gene inactivation to identify host genes required for TTSS- mediated epithelial cell damage. In aim 1,1 will use genome-wide RNAi-mediated gene inactivation to identify host genes required for ExoU or TTSS-mediated cell death. To do this, I will be using D. melanogaster S2 cells, a well-characterized macrophage-like cell line, in combination with an ExoU- mediated cytotoxicity assay. In aim 2, we will test candidate host genes for their requirement in the pathogenesis of PA-mediated lung epithelial damage by using A549 cells, a human alveolar type II cultured cell line. In the future, the role of these gene products can be validated in animal models of disease. [unreadable] [unreadable] [unreadable]